Inflammatory disorders account for a significant number of debilitating diseases. Inflammatory states, such as arthritis, psoriasis, asthma, and possibly atherosclerosis, stem from inflammatory reactions in the joints, skin, and blood vessels. It is generally believed that a central role in the inflammatory reaction is the production of phospholipid metabolites called eicosanoids. The eicosanoids represent a family of important mediators such as the leukotrienes, prostaglandins, lipoxins, hydroxyeicosatetranoic acid, and thromboxanes. It is believed that the generation of eicosanoids is dependent on the availability of arachidonic acid which is liberated from phospholipids by the action of phospholipase A.sub.2 (EC 3.1.1.4).
Phospholipase A.sub.2 (PLA.sub.2) is the common name for phosphatide 2-acylhydrolase, which catalyzes the hydrolysis of the sn-2-acyl ester bond of phosphoglycerides which results in the production of equimolar amounts of lysophospholipids and free fatty acids. see, E. A. Dennis, THE ENZYMES, Vol. 16, Academic Press, New York, (1983). Phospholipase A.sub.2 enzymes are found in all living species and form a diverse family of enzymes. Over eighty phospholipase A.sub.2 enzymes have been structurally characterized, and they show a high degree of sequence homology. J. Chang, et al., Biochemical Pharmacology, 36:2429-2436, (1987); F. F. Davidson and E. A. Dennis, Journal of Molecular Evolution, 31:228-238 (1990).
The best characterized varieties of PLA.sub.2 enzyme are the secreted forms, which are released into the extracellular environment where they aid in the digestion of biological materials. The secreted forms have a molecular weight of about 12-15,000 (Davidson and Dennis, supra). In contrast, cytosolic phospholipases A.sub.2 are found in small amounts within the cell and play a key role in the biosynthetic pathway leading to the formation of the platelet activating factors and the eicosanoids. R. M. Kramer, SIGNAL-ACTIVATED PHOSPHOLIPASES, (M. Liscontdi, ed. 1994) pp. 13-30; J. D. Sharp, et al., Journal of Biological Chemistry, 266:14850-14853 (1991).
The cytosolic phospholipases A.sub.2 have a molecular weight of approximately 85,000 daltons. J. D. Clark, et al., Cell, 65:1043-1051 (1991). Free arachidonic acid is the rate limiting precursor for the production of eicosanoids and is liberated from its membrane phospholipid store by the action of cytosolic PLA.sub.2. E. A. Dennis, Drug Development and Research, 10:205-220, (1987). The same enzymatic step also produces lysophospholipids which may be converted to platelet-activating factors. Thus, it is believed that cytosolic PLA.sub.2 is central to the regulation of the biosynthetic pathways of potent lipid mediators of inflammation.
Recent studies have begun to indicate that a major component of the pathology of Alzheimer's disease is chronic inflammation. See, J. Schnabel, Science, 260:1719-1720 (1993). Indeed, pathological investigations have demonstrated the presence of glial hyperactivity, acute phase proteins, and complement factors within affected areas of the brains of persons affected with Alzheimer's disease. Administration of nonsteroidal anti-inflammatory drugs appears to slow the advance of Alzheimer's disease. Id. Understanding this inflammatory component of Alzheimer's disease, therefore, will lead to advances in novel methods of treating patients suffering from this disease.
Due to the central role in the inflammatory component of Alzheimer's disease that appears to be played by phospholipase A.sub.2, it is desirable to identify and characterize new inhibitors of this enzyme. The present invention provides a novel phospholipase A.sub.2, nucleic acids encoding this enzyme, and assays which may be employed to identify inhibitors having a therapeutic benefit.
Met Gly Ser Ser Glu Val Ser Ile Ile Pro Gly Leu Gln Lys Glu Glu 1 5 10 15 - Lys Ala Ala Val Glu Arg Arg Arg Leu His Val Leu Lys Ala Leu Lys 20 25 30 - Lys Leu Arg Ile Glu Ala Asp Glu Ala Pro Val Val Ala Val Leu Gly 35 40 45 - Ser Gly Gly Gly Leu Arg Ala His Ile Ala Cys Leu Gly Val Leu Ser 50 55 60 - Glu Met Lys Glu Gln Gly Leu Leu Asp Ala Val Thr Tyr Leu Ala Gly 65 70 75 80 - Val Ser Gly Ser Thr Trp Ala Ile Ser Ser Leu Tyr Thr Asn Asp Gly 85 90 95 - Asp Met Glu Ala Leu Glu Ala Asp Leu Lys His Arg Phe Thr Arg Gln 100 105 110 - Glu Trp Asp Leu Ala Lys Ser Leu Gln Lys Thr Ile Gln Ala Ala Arg 115 120 125 - Ser Glu Asn Tyr Ser Leu Thr Asp Phe Trp Ala Tyr Met Val Ile Ser 130 135 140 - Lys Gln Thr Arg Glu Leu Pro Glu Ser His Leu Ser Asn Met Lys Lys 145 150 155 160 - Pro Val Glu Glu Gly Thr Leu Pro Tyr Pro Ile Phe Ala Ala Ile Asp 165 170 175 - Asn Asp Leu Gln Pro Ser Trp Gln Glu Ala Arg Ala Pro Glu Thr Trp 180 185 190 - Phe Glu Phe Thr Pro His His Ala Gly Phe Pro Ala Leu Gly Ala Phe 195 200 205 - Val Ser Ile Thr His Phe Gly Ser Lys Phe Lys Lys Gly Arg Leu Val 210 215 220 - Arg Thr His Pro Glu Arg Asp Leu Thr Phe Leu Arg Gly Leu Trp Gly 225 230 235 240 - Ser Ala Leu Gly Asn Thr Glu Val Ile Arg Glu Tyr Ile Phe Asp Gln 245 250 255 - Leu Arg Asn Leu Thr Leu Lys Gly Leu Trp Arg Arg Ala Val Ala Asn 260 265 270 - Ala Lys Ser Ile Gly His Leu Ile Phe Ala Arg Leu Leu Arg Leu Gln 275 280 285 - Gly Ser Ser Gln Gly Glu His Pro Pro Pro Glu Asp Glu Gly Gly Glu 290 295 300 - Pro Glu His Thr Trp Leu Thr Glu Met Leu Glu Asn Trp Thr Arg Thr 305 310 315 320 - Ser Leu Glu Lys Gln Glu Gln Pro His Glu Asp Pro Glu Arg Lys Gly 325 330 335 - Ser Leu Ser Asn Leu Met Asp Phe Val Lys Lys Thr Gly Ile Cys Ala 340 345 350 - Ser Lys Trp Glu Trp Gly Thr Thr His Asn Phe Leu Tyr Lys His Gly 355 360 365 - Gly Ile Arg Asp Lys Ile Met Ser Ser Arg Lys His Leu His Leu Val 370 375 380 - Asp Ala Gly Leu Ala Ile Asn Thr Pro Phe Pro Leu Val Leu Pro Pro 385 390 395 400 - Thr Arg Glu Val His Leu Ile Leu Ser Phe Asp Phe Ser Ala Gly Asp 405 410 415 - Pro Phe Glu Thr Ile Arg Ala Thr Thr Asp Tyr Cys Arg Arg His Lys 420 425 430 - Ile Pro Phe Pro Gln Val Glu Glu Ala Glu Leu Asp Leu Trp Ser Lys 435 440 445 - Ala Pro Ala Ser Cys Tyr Ile Leu Lys Gly Glu Thr Gly Pro Val Val 450 455 460 - Met His Phe Pro Leu Phe Asn Ile Asp Ala Cys Gly Gly Asp Ile Glu 465 470 475 480 - Ala Trp Ser Asp Thr Tyr Asp Thr Phe Lys Leu Ala Asp Thr Tyr Thr 485 490 495 - Leu Asp Val Val Val Leu Leu Leu Ala Leu Ala Lys Lys Asn Val Arg 500 505 510 - Glu Asn Lys Lys Lys Ile Leu Arg Glu Leu Met Asn Val Ala Gly Leu 515 520 525 - Tyr Tyr Pro Lys Asp Ser Ala Arg Ser Cys Cys Leu Ala 530 535 540
hereinafter referred to as SEQ ID NO:2, and having activity as a phospholipase A.sub.2. This phospholipase A.sub.2 is alternatively referred to as nPLA.sub.2 or PLA.sub.2 -gamma.
The invention also provides an isolated nucleic acid compound that comprises a nucleic acid sequence which encodes the amino acid compounds provided. Particularly this invention provides the isolated nucleic acid compound having the sequence
ACGAGGGGAG GGCTGTTTAA AGGCGCAGGG GCCATTTTAC CTCCAGGTTG GCCCTGCTCA 60 - GGACCAGGAG GAAACACCTC CAGCCCGCGA CCTCCTCCCA CAGGGGGAAA AGGAAAGCAG 120 - GAGGACCACA GAAGCTTTGG CACCGAGGAT CCCCGCAGTC TTCACCCGCG GAGATTCCGG 180 - CTGAAGGAGC TGTCCAGCGA CTACACCGCT AAGCGCAGGG AGCCCAAGCC TCCGCACCGG 240 - ATTCCGGAGC ACAAGCTCCA CCGCGCATGC GCACACGCCC CAGACCCAGG CTCAGGAGGA 300 - CTGAGAATTT TCTGACCGCA GTGCACC ATG GGA AGC TCT GAA GTT TCC ATA 351 Met Gly Ser Ser Glu Val Ser Ile 1 5 - ATT CCT GGG CTC CAG AAA GAA GAA AAG GCG GCC GTG GAG AGA CGA AGA 399 Ile Pro Gly Leu Gln Lys Glu Glu Lys Ala Ala Val Glu Arg Arg Arg 10 15 20 - CTT CAT GTG CTG AAA GCT CTG AAG AAG CTA AGG ATT GAG GCT GAT GAG 447 Leu His Val Leu Lys Ala Leu Lys Lys Leu Arg Ile Glu Ala Asp Glu 25 30 35 40 - GCC CCA GTT GTT GCT GTG CTG GGC TCA GGC GGA GGA CTG CGG GCT CAC 495 Ala Pro Val Val Ala Val Leu Gly Ser Gly Gly Gly Leu Arg Ala His 45 50 55 - ATT GCC TGC CTT GGG GTC CTG AGT GAG ATG AAA GAA CAG GGC CTG TTG 543 Ile Ala Cys Leu Gly Val Leu Ser Glu Met Lys Glu Gln Gly Leu Leu 60 65 70 - GAT GCC GTC ACG TAC CTC GCA GGG GTC TCT GGA TCC ACT TGG GCA ATA 591 Asp Ala Val Thr Tyr Leu Ala Gly Val Ser Gly Ser Thr Trp Ala Ile 75 80 85 - TCT TCT CTC TAC ACC AAT GAT GGT GAC ATG GAA GCT CTC GAG GCT GAC 639 Ser Ser Leu Tyr Thr Asn Asp Gly Asp Met Glu Ala Leu Glu Ala Asp 90 95 100 - CTG AAA CAT CGA TTT ACC CGA CAG GAG TGG GAC TTG GCT AAG AGC CTA 687 Leu Lys His Arg Phe Thr Arg Gln Glu Trp Asp Leu Ala Lys Ser Leu 105 110 115 120 - CAG AAA ACC ATC CAA GCA GCG AGG TCT GAG AAT TAC TCT CTG ACC GAC 735 Gln Lys Thr Ile Gln Ala Ala Arg Ser Glu Asn Tyr Ser Leu Thr Asp 125 130 135 - TTC TGG GCC TAC ATG GTT ATC TCT AAG CAA ACC AGA GAA CTG CCG GAG 783 Phe Trp Ala Tyr Met Val Ile Ser Lys Gln Thr Arg Glu Leu Pro Glu 140 145 150 - TCT CAT TTG TCC AAT ATG AAG AAG CCC GTG GAA GAA GGG ACA CTA CCC 831 Ser His Leu Ser Asn Met Lys Lys Pro Val Glu Glu Gly Thr Leu Pro 155 160 165 - TAC CCG ATA TTT GCA GCC ATT GAC AAT GAC CTG CAA CCT TCC TGG CAG 879 Tyr Pro Ile Phe Ala Ala Ile Asp Asn Asp Leu Gln Pro Ser Trp Gln 170 175 180 - GAG GCA AGA GCA CCA GAG ACC TGG TTC GAG TTC ACC CCT CAC CAC GCT 927 Glu Ala Arg Ala Pro Glu Thr Trp Phe Glu Phe Thr Pro His His Ala 185 190 195 200 - GGC TTC CCT GCA CTG GGG GCC TTT GTT TCC ATA ACC CAC TTC GGA AGC 975 Gly Phe Pro Ala Leu Gly Ala Phe Val Ser Ile Thr His Phe Gly Ser 205 210 215 - AAA TTC AAG AAG GGA AGA CTG GTC AGA ACT CAC CCT GAG AGA GAC CTG 1023 Lys Phe Lys Lys Gly Arg Leu Val Arg Thr His Pro Glu Arg Asp Leu 220 225 230 - ACT TTC CTG AGA GGT TTA TGG GGA AGT GCT CTT GGT AAC ACT GAA GTC 1071 Thr Phe Leu Arg Gly Leu Trp Gly Ser Ala Leu Gly Asn Thr Glu Val 235 240 245 - ATT AGG GAA TAC ATT TTT GAC CAG TTA AGG AAT CTG ACC CTG AAA GGT 1119 Ile Arg Glu Tyr Ile Phe Asp Gln Leu Arg Asn Leu Thr Leu Lys Gly 250 255 260 - TTA TGG AGA AGG GCT GTT GCT AAT GCT AAA AGC ATT GGA CAC CTT ATT 1167 Leu Trp Arg Arg Ala Val Ala Asn Ala Lys Ser Ile Gly His Leu Ile 265 270 275 280 - TTT GCC CGA TTA CTG AGG CTG CAA GAA AGT TCA CAA GGG GAA CAT CCT 1215 Phe Ala Arg Leu Leu Arg Leu Gln Glu Ser Ser Gln Gly Glu His Pro 285 290 295 - CCC CCA GAA GAT GAA GGC GGT GAG CCT GAA CAC ACC TGG CTG ACT GAG 1263 Pro Pro Glu Asp Glu Gly Gly Glu Pro Glu His Thr Trp Leu Thr Glu 300 305 310 - ATG CTC GAG AAT TGG ACC AGG ACC TCC CTG GAA AAG CAG GAG CAG CCC 1311 Met Leu Glu Asn Trp Thr Arg Thr Ser Leu Glu Lys Gln Glu Gln Pro 315 320 325 - CAT GAG GAC CCC GAA AGG AAA GGC TCA CTC AGT AAC TTG ATG GAT TTT 1359 His Glu Asp Pro Glu Arg Lys Gly Ser Leu Ser Asn Leu Met Asp Phe 330 335 340 - GTG AAG AAA ACA GGC ATT TGC GCT TCA AAG TGG GAA TGG GGG ACC ACT 1407 Val Lys Lys Thr Gly Ile Cys Ala Ser Lys Trp Glu Trp Gly Thr Thr 345 350 355 360 - CAC AAC TTC CTG TAC AAA CAC GGT GGC ATC CGG GAC AAG ATA ATG AGC 1455 His Asn Phe Leu Tyr Lys His Gly Gly Ile Arg Asp Lys Ile Met Ser 365 370 375 - AGC CGG AAG CAC CTC CAC CTG GTG GAT GCT GGT TTA GCC ATC AAC ACT 1503 Ser Arg Lys His Leu His Leu Val Asp Ala Gly Leu Ala Ile Asn Thr 380 385 390 - CCC TTC CCA CTC GTG CTG CCC CCG ACG CGG GAG GTT CAC CTC ATC CTC 1551 Pro Phe Pro Leu Val Leu Pro Pro Thr Arg Glu Val His Leu Ile Leu 395 400 405 - TCC TTC GAC TTC AGT GCC GGA GAT CCT TTC GAG ACC ATC CGG GCT ACC 1599 Ser Phe Asp Phe Ser Ala Gly Asp Pro Phe Glu Thr Ile Arg Ala Thr 410 415 420 - ACT GAT TAC TGC CGC CGC CAC AAG ATC CCC TTT CCC CAA GTA GAA GAG 1647 Thr Asp Tyr Cys Arg Arg His Lys Ile Pro Phe Pro Gln Val Glu Glu 425 430 435 440 - GCT GAG CTG GAT TTG TGG TCC AAG GCC CCC GCC AGC TGC TAC ATC CTG 1695 Ala Glu Leu Asp Leu Trp Ser Lys Ala Pro Ala Ser Cys Tyr Ile Leu 445 450 455 - AAA GGA GAA ACT GGA CCA GTG GTG ATG CAT TTT CCC CTG TTC AAC ATA 1743 Lys Gly Glu Thr Gly Pro Val Val Met His Phe Pro Leu Phe Asn Ile 460 465 470 - GAT GCC TGT GGA GGT GAT ATT GAG GCA TGG AGT GAC ACA TAC GAC ACA 1791 Asp Ala Cys Gly Gly Asp Ile Glu Ala Trp Ser Asp Thr Tyr Asp Thr 475 480 485 - TTC AAG CTT GCT GAC ACC TAC ACT CTA GAT GTG GTG GTG CTA CTC TTG 1839 Phe Lys Leu Ala Asp Thr Tyr Thr Leu Asp Val Val Val Leu Leu Leu 490 495 500 - GCA TTA GCC AAG AAG AAT GTC AGG GAA AAC AAG AAG AAG ATC CTT AGA 1887 Ala Leu Ala Lys Lys Asn Val Arg Glu Asn Lys Lys Lys Ile Leu Arg 505 510 515 520 - GAG TTG ATG AAC GTG GCC GGG CTC TAC TAC CCG AAG GAT AGT GCC CGA 1935 Glu Leu Met Asn Val Ala Gly Leu Tyr Tyr Pro Lys Asp Ser Ala Arg 525 530 535 - AGT TGC TGC TTG GCA TAGATGAGCC TCAGCTTCCA GGGCACTGT G GGCCTGTTGG 1990 Ser Cys Cys Leu Ala 540 - TCTACTAGGG CCCTGAAGTC CACCTGGCCT TCCTGTTCTT CACTCCCTTC AGCCACACGC 2050 - TTCATGGCCT TGAGTTCACC TTGGCTGTCC TAACAGGGCC AATCACCAGT GACCAGCTAG 2110 - ACTGTGATTT TGATAGCGTC ATTCAGAAGA AGGCGTCCAA GGAGCTGAAG GTGGTGAAAT 2170 - TTGTCCTGCA GGTCCCTCGG GAGATCCTGG AGCTGGAGCA TGAGTGTCTG ACAATCAGAA 2230 - GCATCATGTC CAATGTCCAG ATGGCCAGAA TGAATGTGAT AGTTCAGACC AATGCCTTCC 2290 - ACTGCTCCTT TATGACTGCA CTTCTAGCCA GTAGCTCTGC ACAAGTTAGC TCTGTAGAAG 2350 - TAAGAACTTG GGCTTAAATC ATGGGCTATC TCTCCACAGC CAAGTGGAGC TCTGAGAATA 2410 - CAACAAGTGC TCAATAAATG CTTGCTGATT GACTGATGGA AAAAAAAAAA AAAAAAAAAA 2470 - AAAAA 2475
hereinafter referred to as SEQ ID NO:1.
The present invention also provides processes for producing a phospholipase enzyme, said process comprising: (a) establishing a culture of the host cell transformed with an nPLA.sub.2 encoding polynucleotide in a suitable culture medium; and (b) isolating said enzyme from said culture. Compositions comprising a peptide made according to such processes are also provided.
The present invention also provides methods for identifying an inhibitor of phospholipase activity, said method comprising: (a) combining a phospholipid, a candidate inhibitor compound, and a composition comprising a protein of the present invention; and (b) observing whether said protein of the present invention cleaves said phospholipid and releases fatty acid thereby. Inhibitors of phospholipase activity identified by such methods, pharmaceutical compositions comprising a therapeutically effective amount of such inhibitors and a pharmaceutically acceptable carrier, and methods of reducing inflammation by administering such pharmaceutical compositions to a mammalian subject are also provided.
Polyclonal and monoclonal antibodies to the peptides of the invention are also provided.